The transversus abdominis plane block is a regional anesthesia technique. Recently, its impact on early chronic pain and the cumulative need of analgesic medication following inguinal hernia repair is being monitored. In terms of effectiveness and patient safety, it remains unclear whether the approach should be conducted preoperatively through ultrasound guidance, or through intraoperative visual guidance.The study at hand aims to provide more evidence on this topic.A monocentric retrospective matched pair analysis was performed. The intraoperative visual guided and ultrasound guided -transversus abdominis plane block prior to inguinal hernia repair in transabdominal preperitoneal technique were consecutively compared in regard to analgesic effectiveness and complication rate. The data of individuals who were operated on from June 2007 to February 2019 were analyzed. The matching criteria were ASA-Score, Gender, Age ( ±6 years), and hernia size .A total of 116 patients were enrolled. Both groups were homogenous in terms of age, gender contribution, body mass index, ASA-Score, hernia type, and size. The pain score at the postoperative anesthesia care unit was lower in the ultrasound-guided-transversus abdominis plane group without being statistically significant (VAS-Score: 0.67 vs.0.84). Patients of the ultrasound-guided-transversus abdominis plane group received significantly less metamizole on the day of operation (1.29 g (0.96) vs. 1.68 g (0.70), p = 0.015).Due to our findings, we assume that the ultrasound-guided-transversus abdominis plane -Block may reduce postoperative pain and analgesic consumption more effectively than the visual-guided-transversus abdominis plane lock. Further prospective clinical trials are mandatory. •The TAP-Block can be performed ultrasound- and visual-guided.•No TAP-Block related complications appeared among our 116 individuals.•Ptients who received the ultrasound-guided transversus abdominis plane block needed significantly less metamizole on the day of operation. It impaSeveral authors demonstrated that subfascial/subcutaneous local infiltration and anesthetic field blocks reduce early postoperative pain, and the need of analgesic medication following open IHR ,5. MoreoRegarding laparoscopic IHR  and total extraperitoneal (TEP) technique), a total of 6 studies involving 1089 patients demonstrated a statistically compelling transversus abdominis plane (TAP) block impact on pain reduction and painkiller consumption . This prIn terms of effectiveness and patient safety, it remains unclear whether the method should be conducted preoperatively through ultrasound-guidance (UG), or intraoperatively through visual-guidance (IVG). Until now no study has been conducted to compare both approaches to each other. We assume that the IVG-TAP block may be more effective. Ultrasound imaging may facilitate a more precise injection.Thus, the study at hand aims to provide more evidence on this topic.2A monocentric retrospective matched pair analysis was performed. The UG-TAP block prior to primary IHR in TAPP technique was compared to the IVG-TAP block in terms of analgesic effectiveness, use, and complication rate. The data of individuals who were consecutively operated on from June 2007 to February 2019, were analyzed. The matching criteria were ASA-Score, Gender, Age ( ±6 years), and hernia size .The data were collected at the xxx hospital xxx (xxx) between April and May 2020. The study was approved by the Ethics Committee of the ‘Ärztekammer xxx’  in September 2019 (Eth-12/19), and conducted in accordance with the ethical standards of the Helsinki Declaration 1975.The study was registered with the German clinical trial registry DRKS (DRKS00021679). No funding has been received. The work has been reported in line with the STROCSS criteria .The study is based on the patients’ data available from their files. The time of their hospital stay has been analyzed. We did not perform a systematic follow-up.The study has been reported in line with the STROCSS criteria .2.1Patients who received an UG-and IVG-TAP block prior to primary laparoscopic IHR in TAPP technique were included. Being over the age of 16 and physical status of I-III based on the American Society of Anesthesiologists criteria were further inclusion criterias.2.2Patients who underwent a conversion to open surgery, being under the age of 16, allergy to local anesthetics and a recurrent hernia were exclusion criterias.2.3The TAP block was performed through preoperative ultrasound and intraoperative visual guidance. In 2015, the VG approach was chosen, because the UG-TAP block appeared to be more time consuming. Before 2015, all patients received an UG-TAP block.2.3.1Under direct visualization using the laparoscope at the beginning of the operation, the TAP block was conducted. While the abdominal cavity was insufflated, the surgeon palpated the lateral border of the rectal sheath, ensuring adequate lateral placement. Next, a 19 G needle was inserted percutaneously above the iliac spine, inferior to the costal margin at the lateral end of the surgical field. The needle was advanced through the internal and external obliques. Laparoscopically, we ensured that the peritoneum was nearly penetrated. We then tried to retract the needle for 2 mm to reach the proper layer. A small amount of local anesthetic was injected between the internal oblique and transversus abdominis. Appropriate dispersal throughout the layer was visually affirmed prior to the injection of the entire extent of local anesthetic. The same procedure was conducted on the contralateral side in the event of a bilateral inguinal hernia. The amount of local anesthetic for unilateral tap block consisted of 75 mg ropivacaine mixed with 20 cc of normal saline. Twice the amount was used in the prospect of a bilateral inguinal hernia. The process took approximately 1 min. A systematic time recording was not carried out.This technique took approximately 1 min (each side). A systematic time recording was not performed.2.3.2After covering and disinfecting the appropriate integumentary field, the UG-TAP block was conducted under anesthesia through ultrasound guidance  using a 22G needle, and a standard lateral approach was performed preceding the operation.The medial head of the ultrasound probe was positioned on the anterior wall at the level of the umbilicus, with adjustments made to obtain optimal images until observation of the rectus abdominis. We then moved the probe anterolaterally to obtain a transverse view of the abdominal layers, scanning the abdominal wall between the iliac crest and the subcostal margin. This allowed for visualization of the external oblique, internal oblique, transversus abdominis, and, most deeply, the peritoneal cavity.This technique took approximately 5–10 min (each side). A systematic time recording was not performed.2.42 to a pressure of 15 mmHg was insufflated. After hernial sac reduction, a polypropylene mesh (Medtronic®) was inserted through the 10-mm port. The mesh was placed and covered the entire myopectineal orifice. The mesh was not fixated. While lowering the intra-abdominal pressure to 8 mm Hg, the inguinal canal was closed by a running dissolvable suture.More than 10 different surgeons conducted the TAPP IHR in this study. Next to an umbilical 10-mmport for the 30° telescope, two 5-mm ports, 5 cm from umbilicus at the lateral borders of the rectus abdominis muscle were inserted. CO2.5Analysis was done using R (ver. 3.6.1). Data was presented as a number (percentages) for nominal, or as a mean ± SD/median (min-max) for metric variables. For the comparison of nominal variables between groups, Fishers exact test was used for metric variables. Normality was tested by using the Shapiro-Wilk test, and hence the T-test or the Wilcoxon-test. A p-value < 0.05 was considered as statistically significant. No corrections for multiple testing was done.2.6With the exception of clavien-dindo-classification (CDC ) and lenThe primary endpoint was the analgesic cumulative need medication (CNM) of opioids. The secondary endpoints were the CNM of metamizole, NSAIDs, and acetaminophen, all relevant complications according to CDC during the hospital stay, operating time, and length of hospital stay. Another secondary endpoint was the pain level in the PACU using the VAS score . Individuals were asked at least once about their pain level in the PACU. In some cases, the patients were asked several times. The mean value was calculated in these cases.All patients received metamizole 8 g/d, or in case of an allergy, ibuprofen 2400 mg/d as a standard analgesic medication.2.7In April 2020, an MS Excel data sheet was provided. This data was imported into R (ver. 3.6.1), and multiple plausibility checks were performed. In May 2020, inconsistencies of the data were resolved.3A total of 116 patients were enrolled .3.1The univariate analysis on matching criteria and BMI is shown in 3.23.33.3.1Patients in the UG-TAP-Block received an average of 10.2 mg (SD 5.09) of morphine and oxycodone on the day of their operation. Individuals of IVG-TAP-Block group received an average of 8.71 mg (SD 5.34) of morphine and oxycodone on the day of their operation (p = 0.134).Patients in the UG-TAP-Block received an average of 0.38 mg (SD 1.46) of piritramide on the day of their operation. Individuals in the IVG-TAP-Block group received an average of 0.71 mg (SD 2.73) of piritramide on the day of their operation  of acetaminophen on the day of their operation (p = 0.322).Patients in the UG-TAP-Block group received an average of 1.29 g (SD 0.96) of metamizole on the day of their operation. Individuals of the IVG-TAP-Block group received an average of 1.68 g (0.70) of metamizole on the day of their operation (p = 0.015).Patients in the UG-TAP-Block group received an average of 11.6 g (SD 79.0) of NSAIDs on the day of their operation. Individuals of the IVG-TAP-Block group received an average of 6.21 g (SD 18.4) of NSAIDs on the day of their operation  in the PACU. Individuals in the IVG-TAP-Block group stated an average pain level of 0.84 (SD 1.42) in the PACU  in the UG-TAP-Block group had no complications (CDC = 0). No patient suffered from a CDC I–V complication. No TAP-Block related major complication has been detected. .A total of 57 patients (98.27%) in the IVG-TAP-Block group had no complications (CDC = 0). In one case, the data was not applied. No patient suffered from a CDC I–V complication. No TAP-Block related major complication has been detected.3.3.5The average LOS was 2.00 days (SD 0) for the UG-TAP-Block group, and 2.02 days (SD 0.13) for IVG-TAP block group  for the UG-TAP-Block group, and 51.94 (SD 24.7) for the IVG-TAP block group  conducted a blinded randomized clinical trial among 30 individuals. The patients in the TAP-Block group (n = 15) suffered from less pain within 24 h after TAPP inguinal hernia repair . MoreoveThe chosen approach may play a role in prevention of TAP block related complications. To our knowledge, at least four TAP block related complications have been reported . In two In general, it has been frequently elaborated that the TAP block may lead to a reduced analgesic use following IHR ,20. To oWe aimed to further investigate the possible superiority of the UG-TAP. Hence, we reviewed the literature for the TAP block use prior to laparoscopic ventral hernia repair. Our search yielded three relevant publications including a matched pair analysis (n = 52) on our own . Two proA systematic time recording has not been carried out. Nevertheless, to our experience, the IVG-TAP block took us no more than 1 min (on each side). In comparison, at least 5 min were needed for the UG-TAP most likely due to covering and disinfecting the integumentary field. Additional time was needed for the ultrasound head covering. From an economic perspective, the time benefit of the IVG-TAP might be important. Also, in terms of material cost, the intraoperative approach appears to be superior in comparison to the UG-TAP .Although a matching of the individuals was performed, the retrospective study design with a lack of a long-term follow-up and the sample size naturally remains a study limitation. A systematic time and cost recording of the TAP block have not been carried out. Our assumptions on that topic have been solely on our experiences. More than 10 different surgeons conducted the TAPP IHR in this study. It increases the bias of the results as well.5Due to our findings, we assume that the UG-TAP-Block may reduce postoperative pain and analgesic consumption more effectively than the VG-TAP-Block. The ultrasound may facilitate a more precise injection of the local anesthetic in the right layer. On the contrary, the IVG-TAP block appeared to be less time and cost consuming.Further prospective clinical trials are necessary to confirm our assumption.CP, NB, JF, GD, NA, PO, UG, KB, SA, HF and MS declare no conflict of interest.No funding has been received.Not commissioned, externally peer reviewed.None.


Silver nanoparticles (AgNPs) are used in many fields of industry and medicine. Despite the well-established antimicrobial activity, AgNPs are foreseen to be used as anticancer drugs due to the unusual feature—inability to induce drug resistance in cancer cells. The aim of the study was to assess biological activity of AgNPs against MDA-MB-436 cells. The cells were derived from triple-negative breast cancer, a type of breast cancer with poor prognosis and is particularly difficult to cure. AgNPs were toxic to MDA-MB-436 cells and the probable mechanism of toxicity was the induction of oxidative stress. These promising effects, giving the opportunity to use AgNPs as an anti-cancer agent should, however, be treated with caution in the light of further results. Namely, the treatment of MDA-MB-436 cells with AgNPs was associated with the increased secretion of several cytokines and chemokines, which were important in breast cancer metastasis. Finally, changes in the actin cytoskeleton of MDA-MB-436 cells under the influence of AgNPs treatment were also observed. According to the World Health Organization, breast cancer is the most commonly diagnosed female cancer in the world and the most common cause of cancer-related deaths among women . An extrDue to their unique physicochemical properties, NPs are widely used not only in oncology but in many fields of science and industry. One of the most well-known and widely used NPs are silver nanoparticles (AgNPs), which have excellent antibacterial, antiviral and antifungal properties, and are successfully used in many commercial products . TherefoThe general toxicity of AgNPs was tested in various in vitro models, both in cells isolated from normal tissues or cancer-derived lines ,13,14. AConsidering the above reports, the aim of our study was to assess the biological activity of AgNPs against MDA-MB-436 triple-negative metastatic human breast cancer cells. We examined the effect of AgNPs on the viability of MDA-MB-436 cells and the level of oxidative stress and apoptosis biomarkers. Next, we measured the effect of AgNPs on the secretion of cytokines, chemokines and acute-phase proteins. Finally, we investigated AgNPs impact on the actin cytoskeleton of MDA-MB-436 cells.MDA-MB-436 cells were treated with 20 nm or 200 nm AgNPs in the concentration range of 10–100 μg/mL, while AgNPs untreated cells (0 μg/mL) served as a control for viability studies. Cell viability was evaluated after 24 and 48 h incubation by the NR and MTT assays. The NR assay showed that 20 and 200 nm AgNPs are toxic to MDA-MB-436 cells, depending on the time of incubation and the NPs concentration. Statistical analysis showed a significant decrease in the viability of MDA-MB-436 cells after treatment with 20 or 200 nm AgNPs at concentrations of 25–100 μg/mL A. The MTThe impact of 20 and 200 nm AgNPs on the formation of MDA and thiols levels in MDA-MB-436 cells was measured after 24 and 48 h incubation. After a 24 h incubation, a statistically significant increase in MDA was observed only when cells were incubated with 50 μg/mL 20 nm AgNPs, however, an upward trend was also observed for 50 μg/mL 200 nm AgNPs. After a 48 h incubation, an increase in the MDA level was statistically significant in all concentrations and sizes tested, except for 10 μg/mL 200 nm AgNPs A. After An analysis using Proteome Profiler Human Apoptosis Array Kit revealed the presence of 16 out of 35 tested proteins involved in apoptosis . AgNPs tAn analysis using the Human Profiler Cytokine Array Kit revealed the presence of 8 of 36 tested proteins that are involved in the inflammatory process . The treThe effect of AgNPs on the secretion of IL-6 was additionally confirmed by the ELISA assay. There was no change in IL-6 levels in AgNPs-treated cells after 24 h incubation. After 48 h incubation of MDA-MB-436 cells with 20 or 200 nm AgNPs, an increase in IL-6 secreted by the cells was observed. Although in cells incubated with both diameters of 50 μg/mL AgNPs the increase reached statistical significance, the observed effect was very slight A. The efThe impact of 20 and 200 nm AgNPs on MDA-MB-436 actin cytoskeleton was also examined using fluorescent microscopy. In AgNPs treated cells, disruption of the actin cytoskeleton was observed in part of cells, which correlates with previously demonstrated cytotoxicity. In addition, changes in cell morphology, such as a decrease in cell adhesion, presence of cells with an elongated shape and the formation of actin branches at the ends of cells, were also noted, especially in cells treated with high (50 μg/mL) concentrations of AgNPs after a 48 h incubation .There are numerous reports indicating that AgNPs affect cell viability. The AgNPs cytotoxicity depends on numerous factors e.g., type of cells and growth media used in the studies, AgNPs size, shape, functionalization, coating, exposure time, concentration, solubility, distribution of particles and aggregation state ,23. TherThe cytotoxicity of AgNPs is primarily associated with the induction of oxidative stress. It has been demonstrated in numerous cellular models that treatment with AgNPs leads to overproduction of reactive oxygen species (ROS) and to inactivation of the antioxidant defense system, which is manifested, for example, by a decrease in the concentration of reduced glutathione ,25. InduTo the best of our knowledge, this is the first time that the cytotoxic effect of AgNPs on MDA-MB-436 cells has been demonstrated. We also did not find any studies that investigated the oxidative stress parameters or apoptosis markers in this experimental model. Other NPs were also not tested in MDA-MB-436 cells but showed cytotoxicity in other TNBC cells . ReturniSince the induction of the inflammatory process may be directly related to ROS production, we investigated the effect of AgNPs on the secretion of cytokines, chemokines and acute phase proteins. AgNPs increased the secretion of several proteins that had a significant role in the process of tumor metastasis . The higIn terms of secreted cytokines, the impact of AgNPs on MDA-MB-436 cells mimics the effects of tumor-associated macrophages (TAMs). TAMs produce multiple growth factors and inflammatory cytokines , each associated with EMT in cancer cells. On the other hand, the CXCL1 and IL–8 gradient produced by AgNPs treated MDA-MB-436 cells may attract myeloid-derived suppressor cells (MDSCs) that preferentially infiltrate the primary tumor site than metastases. The MDSCs could promote EMT-like changes in cancer cells, via multiple pathways . FinallyIn addition to cytotoxicity, oxidative stress parameters, apoptosis and pro-inflammatory markers, the actin cytoskeleton morphology was examined in AgNPs-treated MDA-MB-436 cells. It should be noted that MDA-MB-436 cells have a mixed phenotype, with both epithelial and mesenchymal characteristics. The reorganization of the cytoskeleton observed in MDA-MB-436 cells can potentially lead to a change in cell phenotype to mesenchymal, which may increase their ability to move and migrate . WhereasIn conclusion, we have demonstrated for the first time the impact of AgNPs on human triple-negative breast cancer MDA-MB-436 cells. We have also shown that AgNPs are cytotoxic to cancer cells and the potential mechanism of their toxicity is a disruption of the oxidative balance. Moreover, AgNP-treated MDA-MB-436 cells enter the path of apoptosis. These effects raise hopes for the use of AgNPs as an anti-cancer agent, however, they should be treated cautiously, since we have also shown that AgNP-treated MDA-MB-436 cells developed some anti-apoptotic changes and secrete increased amounts of several pro-inflammatory proteins associated with breast cancer metastasis. AgNPs also led to changes in cells morphology, which may indicate an increased metastatic potential. Therefore, it seems that although the use of nanomaterials in the diagnosis and treatment of cancer is a great hope of modern medicine, we must not forget that the risk associated with exposure to NPs is still insufficiently known, as demonstrated in this paper for AgNPs.3) were prepared by dispersion of 2 mg of AgNPs in 800 µL of distilled water. Particles dispersions were sonicated on ice using a Vibra-Cell 130 probe sonicator . Then, 100 µL of 10x concentrated phosphate-buffered saline and 100 µL of 15% bovine serum albumin solution was added, according to our previous studies [In our studies, commercially available, non-coated AgNPs of nominal size 20 or 200 nm  were used. NPs stock solutions .The studies were conducted on MDA-MB-436 cells—TNBC derived, pleural effusion origin, infiltrating ducal carcinoma and of claudin-low molecular subtype. The cell line was purchased from Cell Lines Service  and cells were grown according to the manufacturer′s instructions in a humidified atmosphere of 95% air and 5% CO4 cells/well in 200 μl of cell culture medium. The cells were incubated overnight, the medium was then replaced with freshly prepared AgNPs solutions. After a suitable time of treatment (24 or 48 h), the medium was aspired, cells were rinsed with 150 µl of PBS and incubated with 100 µl of neutral red (NR) solution (50 μg/mL) for 4 h at 37 °C. Subsequently, the NR solution was removed, cells were washed with 150 µL of PBS and 200 µL of an acetic acid ethanol solution  was added to each well. After 20 min of gentle shaking, NR absorbance was measured spectrophotometrically at 540 nm using the Omega FLUOstar microplate reader .MDA-MB-436 cells were seeded in 96-well microplates  at a density of 2 × 104 cells/well in 100 μl of culture medium. At 24 h after cell seeding, cells were treated with suitable AgNPs solutions. After 24 or 48 h incubation, 15 μL of 3--2,5-diphenyltetrazolium bromide (MTT) of 5 mg/mL stock solution was added to each well and incubated for 3 h at 37 °C. The fluid was then removed and 100 µL of dimethyl sulfoxide (DMSO) was added to each well. After 2 min of intense shaking, optical density was read at 570 nm by the Omega FLUOstar microplate reader .MDA-MB-436 cells were seeded in 96-well microplates  at a density of 1 × 10g for 10 min at 4 °C), the supernatant was discarded and the pellet was washed twice with PBS, then precipitated cells were frozen at −80 °C for further analysis. Cells lysates were prepared by suspending cells pellets in radioimmunoprecipitation assay buffer (RIPA)  supplemented with a protease and phosphatase inhibitors cocktail.The appropriate numbers of MDA-MB-436 cells were seeded on 6-well cell culture plates and left overnight to obtain optimal attachment to the surface. Subsequently, fresh AgNPs stock suspension (or medium without NPs for control plates) was added (10 or 50 μg/mL AgNPs). After 24 or 48 h, medium was aspired, cells were washed with PBS and trypsinized, collected and transferred into Eppendorf tubes. Then, cells were centrifuged  level in lysates of MDA-MB-436 cells treated with 20 and 200 nm AgNPs was determined using a commercially available, based on reaction with thiobarbituric acid, QuantiChromTM TBARS Assay Kit , as recommended by the manufacturer. Cells cultured in medium without the addition of NPs were the control. The spectrophotometric analysis of the reaction product at 535 nm was carried out using an Omega FLUOstar microplate reader .The level of thiol compounds (-SH groups) was determined in lysates of MDA-MB-436 cells treated with 20 and 200 nm AgNPs, using the Fluorometric Thiol Assay Kit . The analysis, based on the Elman′s reaction, was carried out in accordance with the manufacturer’s instructions. Cells cultured in medium without the addition of NPs were the control. The fluorimetric analysis of the reaction product was carried out using an Omega FLUOstar microplate reader .® Bio Max® light film  and the intensity of specific protein points was quantified using commercially available Image J software . The result for protein was presented as the average of two corresponding points, after subtracting the negative control (background intensity) and normalizing the result to the positive control (reference points).The semi-quantitative Proteome Profiler Human Apoptosis Array Kit  assay was used for the determination of the apoptosis markers in MDA-MB-436 cells treated with 50 μg/mL 20 or 200 nm AgNPs for 48 h. AgNPs-treated MDA-MB-436 cells lysates from three independent experiments were mixed together, then used in the Proteome Profiler Human Apoptosis Array Kit, according to the manufacturer’s protocol. In the next step, membranes containing apoptosis-related antibodies were developed on the Carestreamg for 10 min at 4 °C) to remove NPs, and frozen at −80 °C for subsequent enzyme-linked immunosorbent (ELISA) and Human Profiler Cytokine Array Kit assays.MDA-MB-436 cells were seeded on 6-well cell culture plates and left overnight to obtain optimal attachment to the surface. Subsequently, AgNPs stock suspension (or medium without nanoparticles for control plates) was added (10 or 50 μg/mL AgNPs). After incubation for 24 or 48 h, the cell culture medium was collected and centrifuged . Then, the intensity of specific protein points was quantified using commercially available Image J software . The average of two points corresponding to one protein was calculated, background intensity was subtracted (negative control) and protein intensity was normalized to reference points (positive control).For the determination of the protein profile in culture media of MDA-MB-436 cells treated with 50 μg/mL of 20 or 200 nm AgNPs for 48 h, the commercially available Human Profiler Cytokine Array Kit  was used according to the manufacturer′s instructions. Since this is a semi-quantitative assay and three independent replicates were not performed, in order to obtain the most consistent average result, the conditioned media from the MDA-MB-436 cells treated with AgNPs in three independent experiments were mixed together, and this mixture was used in the Human Profiler Cytokine Array Kit assay. For visualization, membranes were developed on the CarestreamIL-6 and IL-8 release was analyzed in the culture media of MDA-MB-436 cells exposed to 20 or 200 nm AgNPs for 24 or 48 h using Human IL-6 ELISA Kit  and Human IL-8 ELISA Kit  following the manufacturer′s protocol. Spectrophotometric analysis of the reaction product was carried out at 450 nm using an Omega FLUOstar microplate reader .® Lab-Tek® Chamber Slide™, Thermo Fisher Scientific Inc., Waltham, MA, USA) overnight, then treated with appropriate AgNPs solutions. After 24 or 48 h incubation, cells were fixed with 3.7% paraformaldehyde and permeabilized with 0.1% Triton X. The presence of actin filaments was evaluated using phalloidin-tetramethylrhodamine B isothiocyanate . The cell nuclei were additionally stained with DAPI . Fluorescence labeling was analyzed under a fluorescent microscope Olympus BX51 .To evaluate 20 and 200 nm AgNPs impact on the actin cytoskeleton of the MDA-MB-436 cells, the cells were grown on 8-well cell culture slides .With the exception of the results of Proteome Profiler Human Cytokine Array and Proteome Profiler Human Apoptosis Array, the obtained results were subjected to statistical analysis using the GraphPad Prism 5.0 software . The data were expressed as mean ± standard deviation. At least three independent experiments were conducted. Statistical significance was evaluated using a one-way analysis of variance (ANOVA). Then, if necessary, Tukey′s test was used for a post-hoc comparison. Differences were defined as statistically significant when the


MYCN amplification that correlates with changes in the expression of miRNAs, which are small non-coding RNA sequences, playing a crucial role in NB development and chemoresistance. In the present study, miRNA expression was analyzed in two human MYCN-amplified NB cell lines, one sensitive (HTLA-230) and one resistant to Etoposide (ER-HTLA), by microarray and RT-qPCR techniques. These analyses showed that miRNA-15a, -16-1, -19b, -218, and -338 were down-regulated in ER-HTLA cells. In order to validate the presence of this down-regulation in vivo, the expression of these miRNAs was analyzed in primary tumors, metastases, and bone marrow of therapy responder and non-responder pediatric patients. Principal component analysis data showed that the expression of miRNA-19b, -218, and -338 influenced metastases, and that the expression levels of all miRNAs analyzed were higher in therapy responders in respect to non-responders. Collectively, these findings suggest that these miRNAs might be involved in the regulation of the drug response, and could be employed for therapeutic purposes.Neuroblastoma (NB) accounts for about 8–10% of pediatric cancers, and the main causes of death are the presence of metastases and the acquisition of chemoresistance. Metastatic NB is characterized by  Neuroblastoma (NB) is an extracranial pediatric tumor originating from the aberrant development of neural crest-derived sympathoadrenal lineage , and is The therapy used in high-risk patients is multimodal, and although the response to treatments is initially positive, subsequently, following the onset of chemoresistance, a large number of patients die as a consequence of relapse and metastasis formation . NB metaMYCN oncogene characterizes the most aggressive high-risk NB subtype [MYCN modulates the expression of several miRNAs [Among the prognostic markers of poor patient outcome, the amplification of the  subtype ,8. More  subtype . Considel miRNAs , the eval miRNAs .Furthermore, since miRNA expression is related to tumor grade, metastasis, and chemoresistance, they could represent a new class of potential therapeutic targets. In this context, we have recently demonstrated in an NB cell line-based model  that EtoMYCN-amplified human stage-IV NB cell line, HTLA-230, was obtained from Dr. L. Raffaghello , while the Etoposide-resistant cell line (ER-HTLA) was selected as previously reported [The reported ,13. CellThe patients included in the study were diagnosed with NB stage M between January 2002 and December 2015. Written consent for the use of samples and clinical data for research was obtained by their legal guardians. The study was approved by the Gaslini Institute Ethical Committee, and all analyses were performed according to the Helsinki declaration.The samples used originated from two groups of patients. With regard to the first group, whole bone marrow (BM) samples were collected in PAXgene™ Blood RNA tubes originating from patients who, after diagnosis, were treated according to the high-risk European protocol. The drugs used in the induction therapy were Cisplatin, Etoposide, Vincristine, Cyclophosphamide, and either Carboplatin or Adriamycin. Patients were divided into two subgroups, responders and non-responders: responders being the patients that could proceed with high-dose chemotherapy and stem cell transplants, and non-responders being the patients who could not proceed and were referred to second-line therapy.With regards to the second group, the samples were represented by tumor specimens containing more than 70% of neoplastic cells and immune-selected metastases from BM samples, as described ,15 and cTotal RNA was extracted from cultured cells using TRIZOL reagent  according to the manufacturer’s instructions. Total RNA (1 μg) was reverse-transcribed into cDNA by a random hexamer primer and SuperScript™ II Reverse Transcriptase .Total RNA and miRNA fractions were extracted from tumor cells and metastases using the miRNeasyMini kit , according to the manufacturer’s protocols. Total RNA and miRNA fractions were extracted from whole BM samples using the PAXgene extraction Kit , according to manufacturer’s protocol. The quality of the RNA fractions was evaluated in the BioAnalyzer 2100 system .http://www.r-project.org using the limma package v.2.14.16 of Bioconductor http://www.bioconductor.org. Only spots with a signal minus background flagged as positive and significant were used in the following analysis as detected spots. Probes with less than 50% of detected spots across all arrays and arrays with a number of detected spots smaller than 50% of all spots on the array were removed. Background corrected intensities of replicated spots on each array were averaged. Data were then log2-transformed and normalized for between-array comparison using quantile normalization [p-values < 0.05 were selected for further analysis. Given the explorative nature of this study, no correction for multiple testing was applied to the screening procedure aimed at selecting multiple sets of microRNAs for subsequent hierarchical clustering analyses. The agglomerative hierarchical clusters, used to detect similarity relationships in microRNA log2-transformed expressions, were computed by the Euclidean distance between single vectors and the Ward method [MiRNA expression profiling was carried out by the Agilent platform following the miRNA Microarray protocol v.3.1.1 . Briefly, 50 ng of total RNA, containing miRNAs and spike-in controls, underwent dephosphorylation and a labeling step with Cyanine 3-pCp. The Cy3-labeled RNA was then purified using the Micro Bio-Spin P-6 Gel Column , and hybridized on human miRNA microarray slides 8×60K  at 55 °C for 20 hours. After washing, the slides were scanned by a G2565CA scanner , and the images were extracted by Feature Extraction software v.10 . Tab-delimited text files were analyzed in R v.2.7.2 software environment d method .Total RNA (10 ng) was reverse transcribed using miR-specific stem-loop RT primers  and components of the High Capacity cDNA Reverse Transcription kit , according to the manufacturer’s protocols. Expression levels of individual miRNAs were detected by subsequent RQ-PCR using TaqMan MicroRNA assays  and a Rotor Gene 3000 PCR System Corbett  with standard thermal cycling conditions, in accordance with manufacturer recommendations. PCR reactions were performed in triplicate in final volumes of 30 µl, including inter-assay controls (IAC) to account for variations between runs. RT-PCR  was used to quantify the expression of has-miR-16, has-miR-15a, has-miR-19b, has-miR-26b, has-miR-27b, has-miR-29c, has-miR-34c, has-miR-126, has-miR-218, has-miR-338, and has-miR-497, according to the manufacturer’s instructions. To normalize the data for quantifying miRNAs, the universal small nuclear RNU38B  as an endogenous control was used [The delta–delta Ct method was employed to calculate the fold change. In brief, each 15 μL of the reaction system contained 0.15 μL of 100 mM dNTPs with dTTP, 1 μL of MultiScribe Reverse Transcriptase (50 U/μL), 1.5 μL of RT buffer (×10), 0.1  μL of RNase inhibitor (20 U/μL), 6.25  μL of nuclease-free water, 5  μL of small RNA, and 3  μL of RT primer. Small RNAs were quantified by a Qubit 3 fluorimeter . Thermal cycling conditions were 30 min at 16 °C, 30 min at 42 °C, and 5 min at 85 °C. Each 20 μL of the reaction system for real-time quantitative PCR contained 1 μL of real-time primer, 1.33  μL of product from the RT reaction, 10  μL of TaqMan Universal PCR Master Mix, and 7.67  μL of nuclease-free water. The reactions were performed in triplicate on a Rotor Gene 3000 PCR System Corbett for 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Along with the Cq values calculated automatically by the SDS software , raw fluorescence data  were exported for further analyses.Array CGH analyses were performed using the Human Genome array-CGH 8 × 60 K Microarray , with an average probe spacing of around 55 Kb.The arrays were performed using Agilent Reference DNAs, analyzed with the Agilent Microarray Scanner Feature Extraction Software version 11.5, and Agilent Genomic Workbench 7.0.4.0 software using the ADM-2 algorithm. Genomic positions of the rearrangements refer to the public UCSC database GRCh37.Principal components analysis (PCA) is a data display method for multivariate data. n variables, the PCA aims to find new directions and linear combinations of the original ones [Given a data set in which each sample is described by nal ones ,20.The first component (PC1) corresponds to the direction explaining the maximum variance, while PC2 is the direction, orthogonal to PC1, explaining the maximum variance not explained by PC1, and so on. The result of such a transformation is that a limited number of components is sufficient to explain the relevant part of the information.The loadings are the coefficients of the linear combinations corresponding to the PCs. By plotting them in a loading plot, it is possible to understand the relationships among the variables in the multivariate space. On the other side, the score plot (the scores being the coordinates of the samples in the new space defined by the PCs) allows the visualization of the location of samples in the space described by the PCs, making it possible to check similarities and differences among the samples. www.gruppochemiometria.it) [The elaborations and the plots were carried out through the software CAT  .Results were expressed as mean ± SEM from at least three independent experiments. The statistical significance of the parametric differences among the sets of experimental data was evaluated by one-way ANOVA and Dunnett’s test for multiple comparisons. Statistical analysis of the mitotic index and reporter assays data was performed using the Fisher’s exact test.In order to identify the miRNAs involved in chemoresistance, miRNA microarray analyses were performed on HTLA-230 and ER-HTLA cells.As shown in p < 0.01 for statistical significance, showed that a total of 35 miRNAs significantly changed their expression, three being up-regulated and 32 down-regulated. The list of these 35 miRNAs is available in the Supplementary Material .For the first time, to our knowledge, this data confers a possible role in NB chemoresistance to miR-27 and miR-218. In fact, despite their expression being found to be down-regulated in several chemoresistant cancers see  48,49,5,73,74,75tatic NB ,69,70, tIn addition, these results confirm the down-regulation of miR-15 and miR-16 in ER-HTLA cells, as found in our previous study . MoreoveSince both genetic and epigenetic mechanisms have been demonstrated to influence NB biology , in ordeMoreover, our data, identifying some chromosomal regions that are more frequently altered in ER-HTLA cells, is in line with the results obtained in a previous paper reporting a gain of 13q14.1-32 and a loss of 17q in other NB chemoresistant cell lines . Since tIn order to evaluate in vivo the expression of miRNAs and their potential role in NB chemoresistance, ten whole BM samples, taken at diagnosis from NB patients either sensitive (responder) or resistant (non-responder) to induction therapy, were randomly selected from our biobank, and six miRNAs from By comparing the expression of miRNAs in therapy-sensitive and therapy-resistant NB patients, only miR-16 was significantly down-regulated in the bone marrow of non-responder patients , while tThese findings, while confirming a potential role of miR-16 in delivering intrinsic chemoresistance of NB, do not confirm the other results obtained from ER-HTLA cells. However, this is not unusual when comparing in vitro with in vivo data, most likely due to the variability found in individual patients. Nevertheless, it should be noted that the content of NB cells in the BM samples ranged from 5% to 35%, making the normal hematopoietic cells prevalent, and thus potentially masking miRNA down-regulation occurring in neoplastic cells.Therefore, in order to better understand the role that these miRNAs could possibly have in NB biology, their expression was tested in ten primary tumors and ten immune-magnetically-enriched NB metastases from stage M NB patients, randomly selected from our biobank. The NB patients’ features of this new set of samples are reported in MYCN-amplified metastases by about 30% as compared to MYCN-amplified tumors, while no significant changes were observed in the expression of the other miRNAs. It is interesting to note that the MYCN status did not influence the expression of these latter miRNAs, neither in tumors nor in metastases  was carried out by collecting miRNA expression profiles analyzed in bone marrows infiltrates, tumors, and metastases. The first PCA has been performed on responder (four) and non-responder (four) patients’ samples. The loading plot showed that all of the variables had similar positive loadings on PC1, meaning that the score on PC1 can be considered as a global quantitative index. On the other hand, PC2 mainly explains the contrast between miR-15, miR-218, and -16 variables , and between miR-15 and -16, which had positive loadings on PC2 (group 2). The two groups of correlated variables were uncorrelated, since their directions from the origin were orthogonal (The presence of chemoresistant cells in the primary tumor and within bone marrow is the most powerful negative prognostic factor for patients with NB. The acquisition of chemoresistance and the ability to metastasize are the results of genetic and epigenetic mechanisms and, among them, miRNAs can play a crucial role . In factIn addition, miR-19b, miR-338, and partially miR-218, whose down-regulation correlates with the metastatic process, could have prognostic value as biomarkers of NB progression.


EEG data was collected during the visual stimulation paradigm, which consisted of four main phases, a pre-tetanus block, photic stimulus, early post-tetanus, and late post-tetanus. The amplitude of the N1b component of the pre-tetanus, early post-tetanus and late post-tetanus visual evoked potentials were calculated. Change in N1b amplitude was calculated by subtracting pre-tetanus N1b amplitude from early and late post-tetanus. Results demonstrated a significant difference between pre-tetanus N1b  and early N1b , t (18) = 2.761, p = 0.039, d = 0.633. No difference was observed between pre-tetanus N1b and late N1b (p = 0.36). In conclusion, our findings suggest that it is possible to induce changes in the amplitude of the visually evoked potential N1b waveform in the visual cortex of humans non-invasively. Additional work is needed to corroborate that the potentiation of the N1b component observed in this study is due to similar mechanisms essential to prolonged strengthened neural connections exhibited in cognitive structures of the brain observed in prior animal research. If so, this will allow for the examination of strengthened neural connectivity and its interaction with multiple human sensory stimuli and behaviors.Sensory systems are widely known to exhibit adaptive mechanisms. Vision is no exception to input dependent changes in its sensitivity. Recent animal work demonstrates enhanced connectivity between neurons in the visual cortex. The purpose of the present experiment was to evaluate a human model that noninvasively alters the amplitude of the N1b component in the visual cortex of humans by means of rapid visual stimulation. Nineteen participants (M The visual cortex has been shown to exhibit input dependent adaptations. Specifically, the human visual cortex has been shown to adapt to color contrast , orientaEvent-related potentials have been found to capture input dependent adaptations. Specifically, the amplitude of the N1 complex has been shown to be influenced by attention . MoreoveIn the present study, written as a brief report, our goal was to noninvasively examine the amplitude of the N1b component in the visual cortex of humans by multiple presentations of rapid visual stimulation. We hypothesized that bilaterally stimulating the human visual cortex with horizontal black and white reversal checkerboards would alter the N1b component of the visual cortex. If effective, this paradigm will provide an understanding of context-specificity of visual adaptation.A total of 19 right-handed college men and women participated in the present study. All subjects had normal or corrected vision. Informed consent was obtained from each participant. Due to the use of rapid visual stimulation, only participants who reported that they did not suffer from any neurological conditions, epilepsy, or migraine headaches were included. All procedures adhered to the rules of the Declaration of Helsinki and was approved by the University of Mississippi ethics committee (#19-047). All participants provided written consent prior to participation.EEG data were collected using a NuAmps 40 channel, 22-bit, digital amplifier , with a fitted 32-channel Quik-Cap with integrated bipolar leads for vertical and horizontal eye movement. Quik-Caps were manufactured of highly elastic breathable Lycra material with soft neoprene electrode gel reservoirs for enhanced patient comfort. All electrodes were placed according to the International 10–20 electrode placement standard . For ther is the size of the stimulus on the screen, and d is the distance of the observer from the screen. The stimuli were presented on a computer monitor (30-inch x flat-screen SVGA monitor with a resolution of 1920 × 1080 pixels Full HD at 60 Hz), 57 cm from subjects, and checkerboard luminance was at 100 percent contrast. Checkerboards were generated by custom software Stim2 (NeuroScan INC) on a Dell Pentium II/200 computer. TTL provided synchronization of stimulus events with EEG acquisition.Visual stimuli consisted of black and white reversal checkerboards presented bilaterally in the visual field. The stimuli subtended 4° of visual angle from the vertical and horizontal midline. The visual angle was computed from the size and the distance of the image from the observer; visual angle calculation: A = (360/2π) (r/d) = 57.3(r/d), where A is the visual angle, pi(π) is approximately 3.14159, See Each pre-tetanus, early post-tetanus, and late post-tetanus blocks lasted 10 min and comprised of interspersed presentations of 420 horizontal black and white reversal checkerboards presented for seventy seconds separated by fifteen seconds of a black screen with no checkerboards. During the pre-tetanus, early post-tetanus, and late post-tetanus blocks, the checkerboards were presented centrally at a rate of 1 Hz (stimulus-onset-asynchrony (SOA) range 950–1120-ms, duration 100-ms). A test frequency of 1 Hz was believed to be low enough not to have a physiological effect and not so long as to unnecessarily prolong the test blocks.The photic tetanus consisted of 1000 consecutive presentations of horizontal black and white reversal checkerboards and lasted for ~2-min. Stimulus duration during the photic tetanus was 100-ms, with a randomly jittered inter-stimulus interval of 67–100-ms .EEG data for each participant was segmented with respect to event markers into 600-ms time epochs (100-ms before and 500-ms after stimulus onset) with epochs averaged by block . Epochs with eye blinks or artifacts were rejected and not included in the averaging process. The amplitude of the N1b component of the pre-tetanus, early post-tetanus, and late post-tetanus visual evoked potentials were averaged for clusters of electrodes  positioned over the left and right posterior aspect of the skull and the occipital cortex, respectively . ClusterA Repeated Measures ANOVA (RM-ANOVA) was utilized to evaluate the changes in amplitude of the N1b component. Main effects for time  were evaluated. Bonferroni-corrected paired t-tests were subsequently employed. Statistical significance was established as a nominal alpha of 0.05. All analyses were computed in JASP (v 0.11.1.0).p = 0.06). However, paired t-test post-hoc comparisons revealed a significant difference between baseline N1b  and early N1b , t(18) = 2.761, p = 0.039, d = 0.633. See t(18) = −0.936, p = 0.362, d = −0.215.The present study replicates that rapid visual stimulation produces changes in the amplitude of the visual evoked potential N1b waveform in the visual cortex measured by EEG, as previously shown . After tThese results support the findings of Teyler et al.  that visIn summary, our replication findings indicate that it is possible to induce changes in the N1b visually evoked potential in the visual cortex of humans non-invasively. We demonstrate this effect using a large, mixed-gender sample. Additional work is needed to corroborate that the potentiation of the N1b component observed in this study is due to similar mechanisms essential to the persistent strengthening of synaptic potentiation in the cognitive structures of the brain observed in prior animal research. If so, this will allow for the examination of increased neural strength and connectivity and its interaction with multiple human sensory stimuli and behaviors.


Anxiety and depression are common in children and adolescents, which can be detected via self-report questionnaires in non-clinical settings like the school environment. Two short versions of the Revised Child Anxiety and Depression Scale  seem to be feasible for administering at schools. The present study evaluated the psychometric properties of the RCADS-25 and RCADS-20 used as screening instruments for anxiety and depression in a general population of schoolchildren and adolescents.The RCADS-25 was completed by 69,487 schoolchildren and adolescents aged 8 to 18. The RCADS-25 and RCADS-20 broad anxiety scales are equal (15 items), but there are two versions of the major depressive disorder (MDD) scale: the RCADS-25 MDD scale (10 items) and the RCADS-20 MDD scale (5 items). The three scales were assessed on structural validity, internal consistency, test-retest reliability, criterion validity, and hypotheses for construct validity.The RCADS-25/RCADS-20 broad anxiety scale demonstrated a sufficient structural validity , internal consistency , test-retest reliability (ICC = 0.73), criterion validity (AUC = 0.79), and all four hypotheses concerning construct validity were confirmed. The RCADS-25 MDD scale demonstrated a sufficient test-retest reliability (ICC = 0.70) and three out of four hypotheses concerning construct validity were confirmed, but its structural validity was suspect . The RCADS-20 MDD scale demonstrated a sufficient structural validity  and internal consistency . Two out of four hypotheses concerning construct validity were confirmed. The test-retest reliability (ICC = 0.60) was insufficient. Since both MDD scales showed shortcomings, the shortening of the RCADS-25 MDD scale was re-examined post hoc by principal component and reliability analyses. The result was an MDD scale with seven items.The RCADS-25/RCADS-20 broad anxiety scale is valid and reliable for screening schoolchildren and adolescents, but the RCADS-25 and RCADS-20 MDD scales showed shortcomings. An MDD scale of seven items showed acceptable psychometric properties. Anxiety and depression are common conditions in children and adolescents and can have a devastating impact on their lives. Prevalence rates vary from 11 to 25% for an anxiety disorder and from 3 to 8% for a depressive disorder . To prevEarly detection of anxious and depressed children and adolescents can be achieved by proactive screening in a non-clinical setting such as the school environment via self-report screening questionnaires , 10. To The Revised Child Anxiety and Depression Scale (RCADS)  seems toN = 1060; age = 6–18) [N = 246–1641; age = 6–18).Previous studies have demonstrated promising psychometric properties of the RCADS-25. The RCADS-25 was developed by shortening five separate anxiety scales of the RCADS-47  to one broad anxiety scale by bi-factor modeling: three items per anxiety scale were selected that loaded strongly on a general broad anxiety factor, strongly and simply on the specific anxiety factors, and were not synonymous . The res = 6–18) . The MDD = 6–18) , 14; ICC = 6–18) ) and con = 6–18)  in schooN = 501; age = 8–14) [N = 544; age = 10–17) [The RCADS-20 has also demonstrated promising psychometric properties in previous research. In the RCADS-20, the broad anxiety scale of the RCADS-25 was left unchanged, but the number of MDD items were reduced by half (five items remaining) in conformance with the MDD scale of the Spanish RCADS-30 , 17. MDD = 8–14) . The MDDDue to the advantage of feasibility of the RCADS short versions in a school setting, the RCADS-25 has been translated into Dutch. In order to be able to use it as a screening instrument for anxiety and depression in schoolchildren and adolescents, it is important to examine whether the promising psychometric properties of the RCADS-25 and the RCADS-20 can be replicated and to add research on the remaining psychometric properties. The present study offers a comprehensive overview of the structural validity, internal consistency, test-retest reliability, criterion validity, and hypotheses testing for construct validity of the RCADS-25 and the RCADS-20 subscales according to the internationally consensus-based COSMIN taxonomy, definitions, and quality criteria of measurement properties –20. The Participants were 70,777 schoolchildren and adolescents aged 8 to 18 who completed the RCADS-25 online at school; 69,487 students completed the RCADS-25 without too many missing values . The coAdditionally, a convenience sample of the above-mentioned schools was approached for extra data collection procedures by youth health care professionals who carried out the screening program at the schools. The selection of schools was based on their screening program planning and on including a variation of urban and rural schools in the present study. If schools granted permission, informed consents and assents for the extra data collection procedures were sent to parents and children or adolescents.The additional data collection procedures varied per school, class, age, and level of education, aiming to obtain maximum variation in the various subsamples for examining different psychometric properties. To examine test-retest reliability, 277 participants (response rate = 77%) completed the RCADS-25 a second time under the same test conditions as 2 to 4 weeks before when completing the RCADS-25 for the first time during the usual screening. To examine criterion validity, 110 other participants (response rate = 17%) had a semi-structured interview after the usual screening . WiThe RCADS-25  is a shop < 0.01 for MDD [The K-SADS-PL , 23 is a for MDD ; B = 4.1 for MDD ).In the present study, three trained interviewers conducted the screening interview and, regardless of the scores on the screening interview, the supplementary interviews of the current episodes of the panic disorder, separation anxiety disorder, avoidant disorder/social phobia, overanxious/generalized anxiety, obsessive-compulsive disorder, and depressive disorders. The three interviewers were blinded to the RCADS-25 scores. They had a Bachelor’s or Master’s degree in Psychology or Psychiatric nursing, and they regularly held peer meetings to give feedback and to promote uniformity in the administration of the interviews. All interviews were audio-recorded; five randomly selected recordings per interviewer were scored a second time by an external fourth trained interviewer to determine the inter-rater agreement. The observed inter-rater agreement of the screening interview ranged from 87 to 100% for the anxiety disorders and was 87% for MDD. The observed inter-rater agreement of derived diagnoses ranged from 87 to 100% for the anxiety disorders and was 100% for the MDD diagnosis.The SCARED-NL  is a selThe Dutch translation of the revised CDI , 27 was Missing data for RCADS-25 scores were handled in accordance with the RCADS-25 Child Version Scoring Program 3.1 . This prPotential selection bias in the additional data collection procedures was examined through multilevel logistic regression analyses. These analyses were adjusted for school and class by means of a random intercepts model. Odds ratios were calculated for children and adolescents who did and did not complete the additional data collection procedures on the one hand, and gender, age group , and scores below and above the 90th percentile of the RCADS-25 scales on the other, since these scale scores were skewed to the right. This is in line with current practice, as children and adolescents scoring above the 90th percentile are invited for further investigation by school nurses and physicians. The 90th percentiles of the RCADS-25 broad anxiety and MDD-10 scale were determined in a national representative sample with respect to gender, age, region, ethnicity, household size, and social class, based on Statistics Netherlands data from 2017   were conducted to examine the unidimensionality of the separate subscales. Unidimensionality refers to the extent that item responses on a scale are driven by the latent trait the scale purports to measure . First, In case of a lack of a one-factor model fit and a sufficient bi-factor model fit, it was examined whether the broad anxiety scale was unidimensional in essence. Essential unidimensionality was examined by calculating the omega hierarchical, which is a statistic that estimates the proportion of variance in raw scores attributable to the general factor ; an omegInternal consistency was assessed by calculating a Cronbach’s alpha per subscale. An alpha equal to or greater than 0.70 was considered as sufficient . HoweverTest-retest reliability was assessed by calculating an intraclass correlation coefficient (ICC) and its 95% confident interval. ICCs were calculated by the use of a single rater, absolute agreement, and a two-way mixed effect model. ICCs of 0.70 or higher were considered as sufficient .Criterion validity was assessed by calculating receiver operating curves (ROCs). One ROC was calculated for the broad anxiety scale in comparison with any anxiety disorder according to the K-SADS-PL . Two ROCs were calculated for the MDD-10 and MDD-5 scales in comparison with depressive disorders according to the K-SADS-PL.Construct validity was assessed by testing four hypotheses based on inferences from previous studies. It was determined a priori that construct validity of the separate subscales would be sufficient if at least three out of four hypotheses were confirmed .Our first hypothesis was that girls have higher mean scores than boys on the broad anxiety scale, MDD-10 scale, and MDD-5 scale , 6. ThisOur second hypothesis was a positive correlation of 0.6 to 0.7 between the broad anxiety scale and the two MDD scales, since anxiety and depression have been found to be comorbid , 45. TheOur third hypothesis was a positive correlation of at least 0.7 between the broad anxiety scale and the SCARED-NL, of at least 0.65 between the MDD-10 scale and the CDI-2, and of at least 0.6 between the MDD-5 and the CDI-2. These correlations were expected, since related constructs are measured, and previous studies have reported comparable results , 17. MorOur fourth hypothesis was a positive correlation of at least 0.6 between the broad anxiety scale and the CDI-2, and between the MDD scales and the SCARED-NL; in addition, these correlations were expected to be lower than the correlations between the broad anxiety scale and the SCARED-NL and between the MDD scales and CDI-2. This hypothesis was based on the fact that slightly different constructs are measured that were expected to correlate highly.All analyses were conducted in SPSS version 21, with the exception of the CFAs, which were performed using the lavaan package in RStudio version 1.1.463, and of the multilevel regression analyses, which were performed in Stata Intercooled 15.Table Table Table Table Of 110 children and adolescents in the criterion validity subsample, 62% had a broad anxiety score below the 90th percentile, 21% had a score between the 90th and 95th percentiles, and 17% had a score above the 95th percentile. With regard to the MDD-10 scale, these percentages were 66, 19, and 16% respectively. According to the K-SADS-PL, 11 participants (10%) had symptoms of one or two anxiety disorders, one (1%) had symptoms of MDD, and one (1%) had symptoms of both an anxiety disorder and MDD. Figure All four hypotheses concerning the broad anxiety scale, three out of four hypotheses concerning the MDD-10 scale, and two out of four hypotheses concerning the MDD-5 scale were confirmed. Girls had higher scores than boys: mean differences were 2.2 (95% CI = 2.1–2.3) on broad anxiety, 1.3 (95% CI = 1.3–1.4) on the MDD-10, and 0.7 (95% CI = 0.7–0.8) on the MDD-5 scale. A positive correlation was found of 0.64 (95% CI = 0.64–0.65) between the broad anxiety and MDD-10 scale, and of 0.64 (95% CI = 0.63–0.64) between the broad anxiety and MDD-5 scale. Table N = 34,644 and N = 34,843 respectively).Since the findings suggested shortcomings of the MDD-10 and MDD-5 scales, post hoc, the possibility to shorten the MDD-10 scale was re-examined so that it meets all criteria. Therefore, the total sample was randomly divided into a training set and a validation set ; 2. a sufficient internal consistency . In addition, in the various subsamples it was examined whether the MDD-7 scale met the following criteria: 3. a sufficient test-retest reliability (ICC ≥ 0.7); 4. a correlation of at least 0.6 between the MDD scale and the CDI-2; 5. a correlation of at least 0.6 between the MDD scale and the SCARED-NL, but lower than between the MDD scale and the CDI-2.The MDD-7 data showed a mediocre one-factor model fit , a sufficient Cronbach’s alpha of 0.78, a sufficient ICC of 0.70, and a correlation with the CDI-2 of r = 0.68; however, the correlation with the SCARED-NL remained the same (r = 0.68). Deleting two items compared to three items downgraded the results of the one-factor model fit and the test-retest reliability to just below sufficient levels. Deleting four items downgraded the test-retest reliability to insufficient levels.The aim of the current study was to examine the psychometric properties of the RCADS-25 and RCADS-20 scales when applied as screening instruments in a non-clinical population of schoolchildren and adolescents.The structural validity, internal consistency, test-retest reliability, and criterion validity of the RCADS-25/RCADS-20 broad anxiety scale all proved sufficient, and the four hypotheses to examine construct validity were confirmed.A bi-factor model fit with the broad anxiety data, as described by Ebesutani and colleagues , was conIn addition to unidimensionality indicators, omega hierarchical and omega total can be regarded as reliability coefficients. Both coefficients were greater than 0.8, and the omega total was even greater than 0.9, which indicates that the broad anxiety scale is highly reliable. The test-retest reliability was sufficient, which indicates that the error variance is small compared to the variance between individuals. Therefore, the broad anxiety scale is applicable to screening purposes.The criterion validity was adequate, with an AUC comparable with previous research . Since aThe psychometric properties of the RCADS-25 MDD-10 and RCADS-20 MDD-5 scales showed mixed results.Concerning the RCADS-25 MDD-10 scale, the test-retest reliability was sufficient and three out of four hypotheses about construct validity were confirmed, but its structural validity was insufficient. One-factor model fit was rejected, because the CFI was too low, and the TLI and RMSEA showed a mediocre fit. Lack of one-factor model fit suggests that other latent constructs are measured in addition to MDD. The test-retest reliability of the MDD-10 scale was sufficient, but lower than previous research has shown . Also, tThe structural validity and internal consistency of the RCADS-20 MDD-5 scale were sufficient, but its test-retest reliability was insufficient and only two out of four hypotheses about construct validity were confirmed . The fewer number of items of the MDD-5 scale improved its structural validity, as model fit with a one-factor model was sufficient. Although the internal consistency remained acceptable, the reduced number of items degraded the test-retest reliability to insufficient. Also, the construct validity was uncertain, since the correlation between the MDD-5 scale and the CDI-2 was just below 0.6 (r = 0.59); still, this correlation seems fairly strong, and 0.6 is just an arbitrary predefined cutoff value. However, this correlation was lower than the correlation between the MDD-5 and the SCARED-NL (r = 0.66).There are some possible explanations for these shortcomings. An equal or higher correlation between the MDD scales and the SCARED-NL than between the MDD scales and the CDI-2 might indicate an inadequate discriminant validity of the MDD scales or of the CDI-2. The latter possibility is mentioned in previous studies , but notTo overcome the shortcomings of both MDD scales, it was re-examined post hoc whether the MDD-10 could be shortened to an MDD scale with sufficient psychometric properties. To shorten the MDD-10 scale, the analyses of Sandín and colleagues  were repN = 110). Due to the low prevalence of MDD a sample of at least 400 children and adolescents is needed for a high sensitivity, a power of at least 80%, and a p-value of less than 0.05 [Limitations of the present study include the limited sample size to examine criterion validity .


Caenorhabditis elegans arrest post-embryonic development at the first larval stage (L1) right after hatching. As arrested L1 larvae, C. elegans become more resistant to diverse stresses, allowing them to survive for several weeks expecting to encounter more favorable conditions. L1 arrested at high densities display an enhanced resistance to starvation, dependent on soluble compounds released beyond hatching and the first day of arrest. Here, we show that this chemical communication also influences recovery after prolonged periods in L1 arrest. Animals at high density recovered faster than animals at low density. We found that the density effect on survival depends on the final effector of the insulin signaling pathway, the transcription factor DAF-16. Moreover, DAF-16 activation was higher at high density, consistent with a lower expression of the insulin-like peptide DAF-28 in the neurons. The improved recovery of animals after arrest at high density depended on soluble compounds present in the media of arrested L1s. In an effort to find the nature of these compounds, we investigated the disaccharide trehalose as putative signaling molecule, since its production is enhanced during L1 arrest and it is able to activate DAF-16. We detected the presence of trehalose in the medium of arrested L1 larvae at a low concentration. The addition of this concentration of trehalose to animals arrested at low density was enough to rescue DAF-28 production and DAF-16 activation to the levels of animals arrested at high density. However, despite activating DAF-16, trehalose was not capable of reversing survival and recovery phenotypes, suggesting the participation of additional signaling molecules. With all, here we describe a molecular mechanism underlying social communication that allows C. elegans to maintain arrested L1 larvae ready to quickly recover as soon as they encounter nutrient sources.In a population, chemical communication determines the response of animals to changing environmental conditions, what leads to an enhanced resistance against stressors. In response to starvation, the nematode  C. elegans has been mainly associated to ascaroside pheromones. These pheromones regulate a variety of social behaviors, including mating attraction, aggregation, foraging or avoidance  by the sensory neurons, the insulin receptor DAF-2 gets activated and triggers a phosphorylation cascade. Low insulin signaling leads to the activation of the main final effector of the pathway, the transcription factor DAF-16, which migrates to the nucleus where it regulates the expression of multiple genes  is a major promoter of cell proliferation and growth in presence of food in Caenorhabditis species with density dependence on starvation survival also show L1 aggregation behavior on plates in the absence of food  I, and MOL273 tps-1(ok373) X; tps-2(ok526) II. We generated MOL273 using routine genetic techniques by crossing VC225 tps-1(ok373) X and RB760 tps-2(ok526) II. To quantify recovery time by luminometry we used the reporter strain MRS387 sevIs1 [Psur-5::luc+::gfp] X  X, PR678 tax-4(p678) III and MOL273 tps-1(ok373) X; tps-2(ok526) II to generate MOL274 daf-6(e1377) X; zIs356 IV, MOL216 tax-4(p678) III; zIs356 IV and MOL271 tps-1(ok373) X; tps-2(ok526) II; zIs356 IV. We used the strain VB1605 svIs69[Pdaf-28::DAF-28::GFP] to quantify DAF-28 expression in the head of the arrested larvae. We crossed this strain with CF1038 to generate MOL224 daf-16(mu86) I; svIs69[Pdaf-28::DAF-28::GFP]. The strains not generated here were obtained from the Caenorhabditis Genetics Center (CGC).We cultured and maintained animals according to standard methods , typical::gfp] X . To deteTo arrest L1 larvae by starvation we first obtained embryos from gravid adults by treatment with alkaline hypochlorite solution. Those embryos were incubated in M9 minimum medium at 20°C with gentle shaking leading to their hatching. These larvae newly hatched in absence of food arrest their development as L1. The density of the worm culture was adjusted to the indicated in every experiment after embryo preparation. Although after hatching the population density may vary from the calculated, we considered it important to adjust immediately after embryo preparation, so that the larvae already hatched at the approximate desired density and the released compounds they encountered are consequently at high or low concentration. For recovery experiments depicted in g and resuspended the animals into clean M9. In parallel, we also centrifuged the controls, but, in these cases, animals were not resuspended in clean M9 but in their original medium.For the experiments in which we washed off the medium where the animals hatched, we spun down twice the samples of arrested L1s during 2 min at 850 × g to collect the supernatant without larvae. We froze this CM at −20°C until the moment of use.To prepare the conditioned medium (CM), we obtained embryos and adjusted the sample density at higher than 50 embryos/μl. The day after, we counted the number of hatched animals so we could adjust the arrested L1s to a final concentration of 50 L1/μl. We then centrifuged them twice during 10 min at 5000 × E. coli OP50-1. Prior to measuring, we sealed the plates with a gas-permeable cover . Then, luminescence of each animal was recorded by a highly sensitive microplate luminometer (Berthold Centro LB960 XS3) for 1 s at intervals of 5 min until animals reached adulthood. Luminometers were kept inside temperature-controlled incubators (Panasonic MIR-154) to maintain a constant 20°C temperature during the experiments. We used sample sizes of more than 20 animas per condition and experiment in, at least, three biological replicates. The luminometry data for each animal was analyzed individually and, for statistics, we used the averages of independent replicates to avoid the inflated N value from using individual animals. The analysis was performed as previously described  described above. At the indicated times, we took aliquots of 18 μl of the suspensions of arrested L1 larvae and we placed them on a microscope slide with 2 μl of 10 mM Levamisole to paralyze the animals. We used a Leica M205 FCA stereo microscope to visualize DAF-16::GFP. Thus, we counted the number of animals with nuclear, cytoplasmatic or intermediate localization of DAF-16 in a population of, at least, 100 animals per sample. With these data, we calculated the percentage of animals with each localization of DAF-16. We performed a minimum of three independent biological replicates. To avoid the artificial translocation of DAF-16 we used a reduced concentration of Levamisole. In the same way, during the visualization the room temperature was kept at 20°C to avoid the possible effect of temperature in DAF-16 activation.We used strains containing a translational GFP reporter of DAF-28 to quantify its expression in the head of the arrested L1 larvae. We took aliquots of 10 μl from suspensions of L1s arrested to the indicated densities after 1 or 5 days of arrest and we placed them on a microscope slide with 1 μl of 10 mM Levamisole to paralyze the animals. We then acquired pictures of these worms using a Leica M205 FCA stereo microscope. We analyzed those pictures with FIJI software, which allowed us to measure the signal intensity of a selected area. We analyzed more than 20 animals per condition and experiment, and we performed a minimum of three individual biological replicates per experiment. Due to discrepancies in the raw data between replicates, we normalized the signal measured for each worm to the average signal of the replicate. We used the averages of the normalized signal per condition and replicate to perform the statistics analysis.g to remove the animals and we collected the supernatant for measurement. We added potassium phosphate buffer pH 6 to the samples to a final concentration of 0.1 M and trehalase enzyme (SIGMA) to 1.5 U/ml. This mix was incubated overnight at 37°C with no shaking. We then added the glucose oxidase/peroxidase mix containing 10 U/ml glucose oxidase, 2 U/ml horseradish peroxidase and 80 μg/ml O-dianisidine. We incubated the samples at 37°C for 30 min, we then added H2SO4 at a final concentration of 4.3 N and we measured the OD of the samples at 540 nm. A standard curve was prepared in parallel with known concentrations of trehalose, so we could transform OD values into trehalose content. For each sample we performed a parallel reaction in which trehalase was substituted by water in order to detect free glucose, what would make us overestimate the content in trehalose. We referred the trehalose content measured to the number of larvae actually hatched for each sample, what we determined the day after egg preparation.Trehalose content in the medium of arrested L1 larvae was measured by enzymatic determination of released glucose after hydrolysis of trehalose by trehalase enzyme, using the glucose oxidase/peroxidase method . We spun down the L1 cultures for 5 min at 5000 × E. coli. This way, the effect of density during arrest is separated from a possible effect of density during recovery or its effect during developmental timing   in a DAF-16 dependent manner, being shorter in a t strain . At the t strain . Arrestet effect , have ans (ILPs) . DAF-28 s (ILPs) . Taking ensities . To do tensities , and theensities . Thus, tensities . With tharvation . At the s tested . The wildaf-16 mutant we could not use such a long starvation period, since at this time arrested L1 larvae of this mutant barely survive. Instead, we analyzed recovery time in the daf-16 mutant after 4 days of starvation, when the rate of recovery is at 85%, indicating a similar situation to the wild-type strain at day 8  inside the aggregate , which can act as agonist or antagonist of the insulin receptor DAF-2 (daf-28 expression is downregulated during starvation or upon application of dauer pheromone (daf-28 expression is regulated by a variety of environmental cues, including molecules in the medium and temperature. So, it appears reasonable that the production of this insulin in the sensory neurons could be regulated by the soluble molecules that mediate communication between animals.The presence of unknown signaling molecules in the supernatant of arrested larvae supports survival in L1s at high density and recovers the short longevity of animals at low densities . Accordior DAF-2 . Positivor DAF-2 . DAF-28 or DAF-2 . DAF-28 or DAF-2 . daf-28 heromone . Furtherheromone , and itsheromone . TherefoC. elegans, trehalose is necessary for desiccation tolerance of the dauer larvae (daf-28 expression by trehalose that we show in daf-28. Indeed, our results (daf-28 regulation leading to DAF-16 inactivation. Further investigation will be needed to elucidate the entry point of trehalose into this regulation. The requirements of DAF-28 for the density effect also remain untested. Since ILPs show functional redundancy, a further investigation of the regulation of other insulins by density is necessary to address this question.In a search for possible communication molecules that could be involved in the effect of density, we tested trehalose. We chose it because it is a molecule whose synthesis is upregulated in arrested L1s  and it ar larvae . Trehalor larvae . This lor larvae . In arrer larvae . Howeverr larvae . To workr larvae . Importar larvae . Internar larvae . This amr larvae . Our mear larvae . That rer larvae . Howeverr larvae . Furtherr larvae . This rer larvae  or when r larvae . In thes results  show thatps-1; tps-2 mutant, which lacks both trehalose synthases (tps mutant we detected about a third of the trehalose detected for the wild type (C. elegans embryos before hatching (C. elegans mothers can adapt the sugar provisioning to the embryos in response to changing environmental conditions (C. elegans has not been identified yet. However, in mammals, a glucose carrier is able to transport trehalose (C. elegans, being the glucose carrier FGT-1 the closest homolog to the trehalose transporter Tret1-1 in Drosophila. Actually, the reduction in the expression of fgt-1 leads to an extended longevity, not additive to the extension displayed in daf-2 and age-1 mutants, meaning that insulin signaling is reduced when fgt-1 expression is down (No trehalose content was previously detected in L1 larvae of the double ynthases . Here, iild type . The treild type . We cannild type . During ild type . Two proild type . Howeverhatching , the funnditions . The connditions . Despiterehalose . This co is down .C. elegans, GUR-3 is the only protein with homology to Gr5a, the trehalose receptor of Drosophila melanogaster (Drosophila, Gr5a is a G protein-coupled gustatory receptor required to activate behavioral and sensory responses to trehalose (Drosophila regulates lifespan and the mutant lacking Gr5a lives shorter (gr5a mutant there is an increased trehalose synthesis in an attempt to compensate for the lack of trehalose sensing (To work as a signaling molecule, trehalose must be sensed by the animals. This process must involve a receptor that senses exogenous trehalose. In nogaster . GUR-3 inogaster . GUR-3 anogaster . In Drosrehalose . Trehalo shorter . Trehalo sensing . This is sensing .tps mutant (Although low concentrations of trehalose activated DAF-16 in our experiments, this response was not sufficient to support starvation survival and recovery of animals arrested at low density . This mas mutant , since tC. elegans.The synthesis of a disaccharide in the absence of food appears to be paradoxical, but trehalose is known to be protective, and as such it acts on arrested L1 larvae, where it supports survival . It alsoAll data generated or analyzed during this study are included in this published article and its .AM-C, MO, LG-D, FR-E, and MR-P carried out the experiments. AM-C, MO, and LG-D analyzed the data and interpreted the results. AM-C, MO, and MA-S designed the study. AM-C and MO wrote the manuscript. All authors read, commented and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ultra-high-performance concrete (UHPC) has been used as an advanced construction material in civil engineering because of its excellent mechanical properties and durability. However, with the depletion of the raw material (river sand) used for preparing UHPC, it is imperative to find a replacement material. Recycled sand is an alternative raw material for preparing UHPC, but it degrades the performance. In this study, we investigated the use of graphene oxide (GO) as an additive for enhancing the properties of UHPC prepared from recycled sand. The primary objective was to investigate the effects of GO on the mechanical properties and durability of the UHPC at different concentrations. Additionally, the impact of the GO additive on the microstructure of the UHPC prepared from recycled sand was analysed at different mixing concentrations. The addition of GO resulted in the following: (1) The porosity of the UHPC prepared from recycled sand was reduced by 4.45–11.35%; (2) the compressive strength, flexural strength, splitting tensile strength, and elastic modulus of the UHPC prepared from recycled sand were enhanced by 8.24–16.83%, 11.26–26.62%, 15.63–29.54%, and 5.84–12.25%, respectively; (3) the resistance of the UHPC to penetration of chloride ions increased, and the freeze–thaw resistance improved; (4) the optimum mixing concentration of GO in the UHPC was determined to be 0.05 wt.%, according to a comprehensive analysis of its effects on the microstructure, mechanical properties, and durability of the UHPC. The findings of this study provide important guidance for the utilisation of recycled sand resources. Ultra-high-performance concrete (UHPC) is a new construction material with excellent mechanical properties and durability , which mThe preparation of concrete using recycled sand has been widely investigated. Kumar et al.  reportedThe use of nanomaterials can improve the properties of the matrix in cement-based materials prepared from recycled sand, enhancing the material performance. With the advancement of nanotechnology, the mixing of nanomaterials into cement-based materials has been increasingly researched. A wide variety of nanomaterials have been investigated, including nano-silicon oxide ,24,25, nIn this study, the properties of UHPC prepared from recycled sand were enhanced via mixing with GO additives. The primary objective was to investigate the effects of GO as an additive on the mechanical properties and durability of recycled-sand-based UHPC (RS-UHPC) at different mixing concentrations. Specifically, the effects of the GO additive on the mechanical properties  and durability (chloride-ion penetration resistance and freeze–thaw resistance) of the RS-UHPC were systematically analysed at three different concentrations. Additionally, the effects of the GO additive on the microstructure of the RS-UHPC were investigated. The results provide important guidance for the application of UHPC made from recycled sand.The following materials were procured to prepare the concrete sample in this study: (1) P·Ⅱ52.5 grade cement produced by Jiangnan-Xiaoyetian Cement Co., Ltd. ; (2) 95# micro-silicon powder produced by Aiken International Trading (Shanghai) Co., Ltd. ; and (3) grade Ⅰ fly ash produced by Zhuhai Minghui Trading Co., Ltd. . The chemical compositions of the cement, silicon powder, and fly ash used in this study are presented in Prior to the preparation of the UHPC, the recycled sand was soaked in water for 12 h, fully saturated with water, and then the recycled sand was washed by water, to remove mud. Eventually, the recycled sand was dried naturally in air at ambient temperature. These procedures ensured that the inside of the recycled sand was saturated with water, while the surface was still dry. Subsequently, an ordinary mixer was used to prepare RS-UHPC. The detailed preparation process was as follows. (1) Cement, silica fume, fly ash, and an expansion agent were mixed in the mixer, with stirring for 4 min. (2) Recycled sand was added, and the stirring was continued for 3 min. (3) Approximately ¾ water was added to the water reducer, and thorough mixing was performed, using a glass rod. The product was then added to the mixture obtained in Step (2), followed by 3 min of stirring. (4) The glass rod was rinsed, along with the container of the water reducer with the remaining water. Then, the entire solution was added to the mixture obtained in Step (3). Stirring was continued for 5 min. (5) Steel fibres were added uniformly to the mixture obtained from Step (5), with stirring for 5 min.Different types of UHPC test pieces were prepared from recycled sand, depending on the objective of the experiment. After the test pieces were moulded, the moulds were wrapped with plastic film and cured for 48 h, at room temperature. Subsequently, the moulds were dissembled, and the RS-UHPC test pieces were further cured in a standard curing room, for 28 days. The temperature and relative humidity of the curing room were maintained at 20 ± 1 °C and >95%, respectively.To examine the microstructure of the RS-UHPC, we measured the porosity and pore-size distribution of RS-UHPC samples, using AutoPore IV mercury intrusion porosimetry (Micromeritics Instrument Corporation). This instrument can measure the pore size in the range of 3.6 nm to 400 μm, with a maximum pressure of 414 MPa.3. There were eight RS-UHPC test pieces for each component mixing ratio. The tensile strength of the RS-UHPC was determined as the average of five experimental measurements. The flexural strength of the RS-UHPC was measured, using a universal testing machine and a four-point flexural experimental device at a loading rate of 0.08 MPa/s. The dimensions of the test piece were 100 × 100 × 400 mm3. There were 10 RS-UHPC test pieces for each component mixing ratio. The flexural strength of the RS-UHPC was determined by taking the average measurements for five splitting-tensile-strength experiments. The splitting tensile strength of the RS-UHPC was measured by using a universal testing machine and a splitting tensile strength experimental device at a loading rate of 0.08 MPa/s. The dimensions of the test piece were 100 × 100 × 100 mm3. Eight RS-UHPC test pieces were prepared for each component mixing ratio. The splitting tensile strength of the RS-UHPC was determined as the average of five experimental measurements. Finally, the elastic modulus of the RS-UHPC was measured, using a universal testing machine and a micro-deformation experimental device at a loading rate of 0.08 MPa/s. The dimensions of the test piece were 100 × 100 × 300 mm3. There were 10 RS-UHPC test pieces for each component mixing ratio. The elastic modulus of the RS-UHPC was determined as the average of six experimental measurements. All four mechanical properties of the RS-UHPC were measured in accordance with the Chinese standard “GB/T, 50081-2002” [The tensile stress of the RS-UHPC was measured by using a universal testing machine at a loading rate of 0.80 MPa/s. The dimensions of the test piece were 100 × 100 × 100 mm81-2002” .3. The mass and dynamic elastic modulus of the test piece were measured prior to the freeze–thaw experiment and every 30 test cycles during the experiment. A total of 10 RS-UHPC test pieces were prepared for the durability test. The experiments were performed five times, and the average values of the measurements were recorded.The chloride-ion penetration resistance and freeze–thaw resistance of the RS-UHPC were analysed in accordance with the Chinese standard “GB/T 50082-2009” . The tesThe porosity of the RS-UHPC samples containing different concentrations of GO is shown in The pore-size distributions of RS-UHPC containing different concentrations of GO are shown in The compressive strengths of the RS-UHPC samples containing different concentrations of GO are presented in The flexural strengths of RS-UHPC samples containing different concentrations of GO are shown in The splitting tensile strengths of RS-UHPC samples containing different concentrations of GO are shown in The elastic moduli of the RS-UHPC samples containing different concentrations of GO are shown in As indicated by the aforementioned results, adding GO to the UHPC sample at a concentration of 0.075 wt.% (higher than the optimum GO content) yielded a slight degradation in the mechanical properties, as compared with the optimum level. This is consistent with the results of Wang et al. . It can −12, 1.08 × 10−12, 1.02 × 10−12, and 1.05 × 10−12 m2 s−1, respectively. Thus, the migration coefficients decreased in the order of RU0 > RU25 > RU75 > RU50. This sequence suggests that the chloride-ion migration coefficient of UHPC does not decrease linearly with the increasing GO concentration. The chloride-ion migration coefficients of RU25, RU50, and RU75 were reduced by 6.90%, 12.07%, and 9.48%, respectively, compared with that of RU0. Therefore, adding GO to RS-UHPC can significantly improve its chloride-ion penetration resistance. This finding is consistent with the results of Guo et al. [The chloride-ion penetration resistance of RS-UHPC samples containing different concentrations of GO is shown in o et al. . The chlo et al. . Becauseo et al.  and imprThe mass loss rates of RS-UHPC samples containing different concentrations of GO are shown in The relative dynamic elastic moduli of the RS-UHPC samples containing different concentrations of GO are shown in According to the previously reported criterion , the freThe chloride-ion penetration resistance and freeze–thaw resistance of the RS-UHPC were both degraded slightly when GO was mixed in the UHPC at a concentration of 0.075 wt.% (exceeding the optimum concentration). This performance degradation is attributed to the same factors responsible for the degradation of the mechanical properties, i.e., adding GO to UHPC at a high concentration results in the agglomeration of GO.In summary, the RS-UHPC samples prepared in this study had excellent mechanical properties and durability. According to a comprehensive analysis of the microstructure, mechanical properties, and durability of UHPC, the optimum concentration of GO to be mixed in UHPC was determined to be 0.05 wt.%. The results indicated that adding GO can enhance the pore structure of RS-UHPC, which was a major factor contributing to the improved mechanical properties and durability of the RS-UHPC prepared in this study.It should be highlighted that the mechanical properties and durability of RS-UHPC were significantly enhanced due to the incorporation of a small quantity of GO, because the degree of cement hydration of RS-UHPC was increased, and the pore structure of RS-UHPC was improved. However, the mechanical properties and durability of RS-UHPC were slightly decreased, when the GO exceeded the optimum concentration, because of the poor ability of GO to be dispersed in RS-UHPC. The optimum concentration of GO to be mixed in RS-UHPC was determined to be 0.05 wt.%, considering the effects of GO on the microstructure, mechanical properties, and durability of RS-UHPC.A systematic study was performed to analyse the effects of GO additives on the mechanical properties and durability of RS-UHPC at different mixing concentrations. Furthermore, the effects of GO on the microstructure of RS-UHPC were investigated. According to the results, the following major conclusions are drawn.(1)The porosity of the RS-UHPC ranged between 2.19% and 2.47%. Adding GO to the RS-UHPC reduced the porosity by 4.45–11.34%. The most probable pore size of the RS-UHPC was reduced with the GO addition. These findings indicate that adding GO to RS-UHPC can improve its pore structure.(2)The tensile strength of the RS-UHPC ranged between 156.21 and 182.50 MPa. Adding GO to the RS-UHPC enhanced its tensile strength by 8.24–16.83%. This result indicates that adding GO to RS-UHPC can improve its tensile strength.(3)The flexural strength of the RS-UHPC ranged between 15.89 and 20.12 MPa. Adding GO to the RS-UHPC enhanced its flexural strength by 11.26–26.62%. This result suggests that adding GO to RS-UHPC can improve its flexural strength.(4)The splitting tensile strength of the RS-UHPC ranged between 13.37 and 17.32 MPa. Adding GO to the RS-UHPC enhanced its splitting tensile strength by 15.63–29.54%. This result indicates that adding GO to RS-UHPC can improve its splitting tensile strength.(5)The elastic modulus of the RS-UHPC ranged between 41.96 and 47.10 GPa. Adding GO to RS-UHPC increased its elastic modulus by 5.84–12.25%. This result suggests that adding GO to RS-UHPC can enhance its resistance to elastic deformation.(6)−12 and 1.16 × 10−12 m2·s−1. Adding GO to the RS-UHPC reduced its chloride-ion migration coefficient by 6.90–12.07%. This result indicates that adding GO to RS-UHPC can improve its resistance to chloride-ion penetration.The chloride-ion migration coefficient of the RS-UHPC ranged between 1.02 × 10(7)The mass loss rate and relative dynamic elastic modulus of the RS-UHPC after 300 freeze–thaw test cycles were approximately 0.44–0.79% and 95.85–97.38%, respectively. These results indicate that adding GO to RS-UHPC can improve its freeze–thaw resistance.(8)According to the comprehensive analysis of the effects of GO on the microstructure, mechanical properties, and durability of RS-UHPC, the optimum concentration of GO to be mixed in RS-UHPC was determined to be 0.05 wt.%.